A number of approaches have been used to try to increase the number of proteins identified by 2DE analysis. Some researchers have prefractionated starting material into organelle specific fractions. This can be either a simple two-part fractionation such as membrane bound and non-membrane bound proteins, or more complicated separations using ultra centrifugation to separate out specific organelles. These methods require expensive equipment (an ultra centrifuge), and only enrich proteins of a given organelle rather than truly improving resolution. These methods may be useful in specifically investigating the expression patterns of a given organelle or compartment, but may be too cumbersome to be applied in looking at all of the proteins in the proteome.
According to another prefractionation methodology, differential solubility may be used to separate proteins into fractions. Raw material may be sequentially processed in buffers with progressively stronger detergents, and chaotropic agents, to separate proteins based upon their hydrophobic/hydrophilic characteristics. Unfortunately, such methods result in a lot of proteins being present in more than one fraction which increases the difficulty of analysis. Other methods use other electrophoretic techniques such as preparative isoelectric focusing or preparative electrophoresis to prefractionate raw material into discrete fractions. These also require special relatively expensive equipment, and may be time consuming. Use of a liquid phase isoelectric focusing apparatus has been the most vigorously pursued of the electrophoretic approaches. Devices in commercial production such as Gradiflow (Gradipore-Frenchs Forest, NSW, Australia) or Rotofor Cell (Biorad) have been applied to this field but require fairly large volumes, and have difficulties with identifying proteins with isoelectric points near their pI cut offs.